Laser microdissection device

ABSTRACT

The invention relates to a laser microdissection device comprised of a microscope table ( 1 ), which supports a specimen ( 3 ) to be dissected, of an incident lighting device ( 7 ), a laser light source ( 5 ) and of an objective ( 10 ) for focussing the laser beam ( 18 ) of the laser light source ( 5 ) onto the specimen ( 3 ). According to the invention, the microscope table ( 1 ) is not moved during the dissecting process. A laser scanning device ( 9 ) is arranged in the incident lighting device ( 7 ), is comprised of two thick glass wedge plates ( 11   a   , 11   b ), which are tilted toward the optical axis ( 8 ) and can be rotated independently of one another around said optical axis ( 8 ). In addition to the beam deviation caused by the wedge angle of the wedge plates ( 11   a,    11   b ), a beam offset of the laser beam ( 18 ) is produced by the thickness and the tilt of the wedge plates ( 11   a,    11   b ). When both wedge plates ( 11   a,    11   b ) are rotated, the beam deviation and the beam offset of the laser beam ( 18 ) are varied in such a manner that the laser beam ( 18 ) always passes through the middle of the objective pupil ( 19 ) and, at the same time, the beam is guided over the specimen ( 3 ) to be dissected by the beam deviation of the laser beam ( 18 ).

[0001] The invention relates to a laser microdissection device havingthe features of the preamble of independent claim 1.

[0002] Known devices for laser microdissection comprise a direct-lightinstrument, into whose beam path a UV laser beam is introduced. The UVlaser light is guided through the direct-light beam path and focused, bya microscope objective, onto a preparation which is placed on amicroscope stage (scanning stage) that can be displaced in a motorizedfashion. The high energy density produced at the focus by the UV laserlight is used for cutting (=dissection) in the preparation. A cuttingline is obtained by displacement of the microscope stage during cutting,in order to move the preparation relative to the laser beam, which isstatic. Operation is generally carried out using pulsed lasers. In thiscase, a laser pulse produces a small hole in the preparation. A cuttingline is obtained by appropriate sequencing of such holes. To that end,in particular in the case of high-magnification objectives, themicroscope stage must have a high positioning accuracy in order to makeprecise cuts possible. Such microscope stages are expensive.

[0003] When the preparation is moved around the cutting point of thelaser beam, the observer also sees the picture move. This isparticularly problematic when observation is being carried out using aslow camera and a monitor. The monitor picture then becomes blurred andexhibits abrupt changes. It would therefore be more favorable for theuser if the microscope stage, and therefore the preparation, couldremain static during cutting.

[0004] If the microscope stage is static, however, then the laser beamneeds to be moved over the preparation, which is static. So that thelaser beam can be guided over a particular field on the preparation, thelaser beam incident in the objective needs to enter the objective pupilat varying angles. This angle variation needs to be carried out by meansof a scanning instrument in the x and y directions.

[0005] Examples of such scanning instruments are mirror scanners,galvanometer scanners and stepper-motor scanners, as are used inscanning optical microscopes. The scanning instrument in each case needsto be arranged in a plane conjugate with the objective pupil. To thatend, so-called pupil imaging is necessary since the deflected beam wouldnot otherwise strike the objective pupil.

[0006] The disadvantage of these known scanning instruments is that theyrequire such pupil imaging. In the case of microdissection using UVlaser light, UV-compatible pupil imaging would be necessary. In anarrangement with pupil imaging, a series of functional units, forexample the aperture-limitation instrument, the offset optics comprisingtwo displaceable lenses, special gray filters etc., need to be arrangedbetween the scanning instrument and the laser. Such a system thereforeamounts to a large overall length and takes up a great deal of space.Known scanning instruments, including the drive electronics, arefurthermore very expensive.

[0007] It is therefore an object of the present invention to provide acompact, simply constructed and inexpensive laser microdissectiondevice, which does not require any pupil imaging and avoids thedisadvantages of the prior art.

[0008] This object is achieved by a laser microdissection device, having

[0009] a microscope stage, which carries a preparation to be cut,

[0010] and a direct-light instrument, a laser light source and amicroscope objective for focusing the laser light of the laser lightsource onto the preparation,

[0011] which has the following novel features:

[0012] a) that the microscope stage is arranged static with respect tothe x-y direction during cutting,

[0013] b) and that the direct-light instrument contains a laser scanninginstrument, which consists of two thick glass wedge plates which areinclined in relation to the optical axis and can be rotatedindependently of one another about the optical axis, and which produce abeam deflection owing to their wedge angle, the resulting deflectionangle α of the laser beam in relation to the optical axis being variableby rotating the glass wedge plates,

[0014] c) and that the laser beam has a lateral beam offset in relationto the optical axis at the output of the scanning instrument, owing tothe thickness and the oblique setting of the glass wedge plates, and itstrikes the middle of the objective pupil of the objective for alldeflection angles α.

[0015] The technical characteristic involves the configuration andspatial arrangement of the two wedge plates.

[0016] Optical units which can be used to produce a beam deflection arein fact already known. For instance, it would be conceivable to obtain abeam deflection of the laser beam in the region of the direct-lightinstrument by passing the laser beam through either an optical unitcomprising two mutually displaceable lenses (so-called Abat wedge) or anoptical unit comprising two mutually rotatable thin glass wedges. Theseoptical units have the disadvantage, however, that the laser beamexperiences only one beam deflection and then arrives outside the pupilof the objective. It therefore no longer reaches the preparation to becut. Arrangements having said optical units are therefore not suitablefor use as a laser scanning instrument.

[0017] A laser microdissection device according to the inventiontherefore has, in the laser beam path, a laser scanning instrument whichconsists of two thick glass wedge plates. The two glass wedge platesmay, for example, have the same wedge angle and different thicknesses aswell as different inclinations in relation to the optical axis. Otherembodiments of the two wedge plates are conceivable.

[0018] Owing to its wedge angle, as is known, each wedge plate producesa component for the overall beam deflection of the laser beam. (The termwedge angle refers to the angle difference between the front externalsurface and the back external surface of a glass wedge plate.) The twocomponents of the overall beam deflection are added togethervectorially. As a result of mutually independent rotation of the twowedge plates about the optical axis, the directions of the twocomponents of the beam deflection are changed. The two components of thebeam deflection are added together vectorially to give an overall beamdeflection with a deflection angle α of the laser beam in relation tothe optical axis. By means of this, the beam deflection produced overallfor the laser beam is varied in such a way that the laser beam is guidedover the preparation to be cut.

[0019] The rotation of the glass wedge plates also causes a change inthe beam offset at the output of the scanning instrument. This change inthe beam offset compensates for the lateral shift of the laser beam,which is produced in the plane of the objective pupil by the beamdeflection. Consequently, the laser beam always passes centrally throughthe pupil of the objective without variation—irrespective of thedeflection angle α that is produced.

[0020] Through suitable driving of the rotational movement of the wedgeplates, it is possible to produce any deflection angle α and thereforecutting lines of any shape. A parallel setting of the two wedge platesleads to a maximum deflection angle α, while an antiparallel arrangementleads to a deflection angle α=0 (i.e. the laser beam strikes thepreparation along the optical axis). Advantageously, the wedge angles ofthe two wedge plates are selected to be so large that the laser beam isdeviated as far as the edge of the field of view when the deflectionangle α is maximum.

[0021] With the laser microdissection device according to the invention,it is possible to leave the preparation to be cut in a fixed positionand to move the laser cutting point over the preparation with littletechnical outlay. At the same time, the inventive structure of the laserscanning instrument comprising two thick, inclined, rotatable wedgeplates is substantially simpler and less expensive than known beamscanners. In the laser microdissection device according to theinvention, an expensive motorized xy stage (scanning stage) may beobviated, since the cutting quality is not dependent on the positioningaccuracy of the microscope stage. A laser in the ultraviolet (UV) orinfrared (IR) or visible (VIS) spectral range may be used as the laserlight source.

[0022] When the laser beam deflected by the laser scanning instrumentstrikes an objective, then for all objective magnifications, as isknown, the deviation in the object is proportional to the objectivemagnification. If the maximum deflection angle α is precisely so largethat the laser beam is deflected as far as the edge of the field ofview, then this is true for all objectives irrespective of theirmagnification. This means that the spatial resolution of the laserscanning instrument in the field of view is the same for all objectives.In order to pursue a cutting line over the entire field of view, thesame angle settings of the two wedges are successively adopted for allobjectives.

[0023] This is the great advantage of the laser microdissection deviceaccording to the invention, having the described laser scanninginstrument, over a previously known laser microdissection device whichoperates with a static laser beam and an xy stage that is moved. In thecase of an xy stage that is moved, its positioning needs to be carriedout ever-more accurately as the objective magnification increases, andtherefore as the cutting width in the object decreases.

[0024] In contrast to this, in the case of the laser scanning instrumentaccording to the invention, a given angle resolution of the wedgerotation for weak objectives with a larger cutting width automaticallyleads to a larger step size in the object than for strong objectiveswith a small cutting width.

[0025] The fact that the microscope stage is static during the cuttingprocess also has the advantage that the user can observe and control thepreparation during the cutting process. For instance, he or she canalready select the next desired cutting line at the same time as acutting process is taking place.

[0026] A further advantage of the invention is that no pupil imaging isrequired and all the functional units, for example theaperture-limitation instrument, the offset optics comprising twodisplaceable lenses, special gray filters etc., can be integrated in acompact microscope beam path. The device according to the inventiontherefore has a very compact structure.

[0027] In an advantageous embodiment of the laser microdissection deviceaccording to the invention, the rotation of the glass wedge plates iscarried out in a motorized fashion. To that end, each glass wedge plateis assigned a motor, for example a stepper motor, for rotating the glasswedge plate about the optical axis. The motors receive their controlsignals from a motor controller. The positioning accuracy of the steppermotors, which are most favorably driven in micro-step operation, thenalso determines the positioning accuracy of the laser beam on thepreparation.

[0028] In another advantageous embodiment of the laser microdissectiondevice according to the invention, the rotation of the glass wedgeplates is likewise carried out in a motorized fashion. A computer havinga mouse and a monitor is additionally provided. The computer isconnected to the motor controller and to the laser light source. Acamera is furthermore provided which takes a picture of the preparation,said picture being displayed on the monitor. When this embodiment isused, it is possible to produce a laser cut in the preparation bycarrying out the following method steps:

[0029] a) defining a cutting line on the monitor by means of the mouse,

[0030] b) computerized division of the cutting line into a series ofcontiguous cutting holes, the centers of which correspond to thesetpoint positions of the laser beam that are to be occupied on thepreparation during the cutting process,

[0031] c) calculating the deflection angle α of the laser beam for eachindividual position to be occupied, and calculating the associatedrotational settings of the glass wedge plates,

[0032] d) producing the control signals for the motorized rotation ofthe glass wedge plates,

[0033] e) and producing the defined cutting line by deflecting the laserbeam into the calculated setpoint positions by rotating the glass wedgeplates.

[0034] Since the described laser scanning instrument permits very exactguidance of the deflected laser beam, other uses of the lasermicrodissection device according to the invention are also possible. Forexample, the laser beam deflected by the laser scanning instrument maybe used for processing of materials.

[0035] In another form of use, the deflected laser beam is guided undercomputer control, and it is used for scribing surfaces.

[0036] In a third form of use of the laser microdissection deviceaccording to the invention, the deflected laser beam is used as opticaltweezers, by using it to pick up and transfer individual particles.

[0037] The invention will be explained in more detail with reference toan exemplary embodiment with the aid of the schematic drawing, in which:

[0038]FIG. 1 shows a laser microdissection device according to theinvention;

[0039]FIG. 2a shows the beam profile of the laser beam when the glasswedge plates are set parallel;

[0040]FIG. 2b shows the beam profile of the laser beam when the glasswedge plates are set antiparallel.

[0041] In the various figures, equivalent components are denoted by thesame reference numerals.

[0042]FIG. 1 shows a laser microdissection device according to theinvention. The laser microdissection device has a microscope stage 1, onwhich a preparation holder 2 that carries an object support 3 a isarranged, a preparation 3 to be cut being located on the lower side ofsaid object support 3 a. A condenser 4, through which the preparation 3is illuminated, is arranged under the microscope stage 1. During thecutting process, which will be described below, the microscope stage 1is not displaced horizontally, that is to say in the x direction and inthe y direction.

[0043] A laser beam is emitted by a laser light source 5, which is hereconfigured as a UV laser light source, and it is introduced via a firstdeflecting mirror 6 a into a direct-light instrument 7 having an opticalaxis 8. A laser scanning instrument 9 is arranged in the direct-lightinstrument 7. The laser beam passes through the laser scanninginstrument 9 and, via a second deflecting mirror 6 b, it reaches anobjective 10 which focuses the laser beam onto the preparation 3. Thedeflecting mirror 6 b is advantageously configured as a dichromaticsplitter through which an imaging beam path 20, emerging from thepreparation 3 through the objective 10, reaches a tube 22 and eyepieces24.

[0044] The laser scanning instrument 9 consists of two thick glass wedgeplates 11 a, 11 b, which are inclined in relation to the optical axis 8and can be rotated independently of one another about the optical axis8. To that end, the wedge plates 11 a, 11 b are mounted using ballbearings 12. The wedge plate 11 a is firmly connected to a toothed wheel13 a, and the wedge plate 11 b is firmly connected to a toothed wheel 13b. The wedge plates 11 a, 11 b are rotated by means of two assignedstepper motors 14 a, 14 b, the stepper motor 14 a engaging with thetoothed wheel 13 a and the stepper motor 14 b engaging with the toothedwheel 13 b.

[0045] The two stepper motors 14 a, 14 b are connected to astepper-motor control unit 15, which delivers the control signals fordriving the two stepper motors 14 a, 14 b. The stepper-motor controlleris connected to a computer 26, which has a monitor 28 attached to it.The picture of the preparation 3, taken by a camera 16, is displayed onthe monitor 28. A cutting line can be defined on the monitor 28 by meansof a computer mouse (not shown). The computer 26 is furthermoreconnected to the laser light source 5 and supplies the latter withtrigger signals for initiating laser pulses when the glass wedge plates11 a, b have been brought into the setpoint position for the cuttingline by the stepper motors 14 a, b.

[0046] When the two glass wedge plates 11 a, 11 b are rotated, the laserbeam appears at the output of the laser scanning instrument 9 withvarious deflection angles and passes through the objective 10, in eachcase through the middle of the objective pupil. The laser beam can inthis case be guided, by varying the deflection angle, to any positionson the preparation 3 which lie within the field of view of the objective10. By suitably driving the rotation of the two glass wedge plates 11 a,11 b, a cutting line can be produced on the preparation 3. The part ofthe preparation 3 which is cut out falls through the frame-shapedopening in the preparation holder 2 into a collection vessel 17, whichis arranged below the preparation 3 on the microscope stage 1.

[0047] The beam profile of the laser beam in the laser scanninginstrument 9 is illustrated in FIGS. 2a and 2 b. The schematicarrangement of two glass wedge plates 11 a, 11 b in a laser scanninginstrument 9 is shown. In FIG. 2b, for illustration, the wedge angle βof one of the two glass wedge plates (11 a, 11 b) is represented. Thewedge angle β refers to the angle difference between the front externalsurface and the back external surface of the glass wedge plate (11 a, 11b).

[0048] A laser beam 18, which is directed at the wedge plates 11 a, 11b, is emitted along an optical axis 8 from a laser light source 5. Ateach glass wedge plate 11 a, 11 b, owing to the respective wedge angleof the latter, the laser beam 18 experiences a beam deflection. After ithas passed through both glass wedge plates 11 a, 11 b, a deflectionangle α is therefore obtained overall.

[0049] In addition, owing to the thickness and the inclination of theglass wedge plates 11 a, 11 b, a beam offset of the laser beam 18 isproduced at each of the two glass wedge plates 11 a, 11 b. Consequently,after it has passed through both glass wedge plates 11 a, 11 b, thelaser beam 18 has, overall, a beam offset Δ which is so large that thelaser beam 18 always passes through the middle of an objective pupil 19of an objective 10 (not shown in detail).

[0050]FIG. 2a shows the two glass wedge plates 11 a, 11 b in a parallelsetting. In this case, the largest deflection angle α and the largestbeam offset Δ are produced. FIG. 2b shows the two glass wedge plates 11a, 11 b in an antiparallel setting. No beam deflection, i.e. adeflection angle α=0, and no beam offset, i.e. Δ=0, are produced in thiscase.

[0051] For all deflection angles α, the beam offset Δ is precisely solarge that it exactly compensates for the lateral shift of the deflectedbeam in the pupil plane, so that the laser beam strikes the middle ofthe objective pupil 19 for all deflection angles Δ.

[0052] By rotating the two wedge plates 11 a, 11 b about the opticalaxis 8, the beam deflection and the beam offset of the laser beam 18 arevaried in such a way that, for all deflection angles α that are set, thelaser beam 18 always passes centrally through the objective pupil 19, sothat the laser beam 18 is guided over the preparation 3 to be cut.

[0053] The present invention has been described with reference toexemplary embodiments, but it is obvious to any skilled person active inthis technical field that modifications and amendments may be madewithout thereby departing from the scope of protection of the followingclaims. List of references  1 microscope stage  2 preparation holder  3preparation  3a object support  4 condenser  5 laser light source  6deflecting mirrors a, b  7 direct-light instrument  8 optical axis  9laser scanning instrument 10 objective 11 glass wedge plates (11a, 11b)12 ball bearing 13 toothed wheels (13a, 13)b 14 stepper motors (14a,14b) 15 motor controller 16 camera 17 collection vessel 18 laser beam 19objective pupil 20 imaging beam path 22 tube 24 eyepiece 26 computer 28monitor α deflection angle β wedge angle Δ beam offset

1. A laser microdissection device, having a microscope stage (1), whichcarries a preparation (3) to be cut, and a direct-light instrument (7)having an optical axis (8), a laser light source (5) for producing alaser beam (18) and a microscope objective (10) for focusing the laserbeam (18) onto the preparation (3), characterized a) in that themicroscope stage (1) is arranged static with respect to the x directionand the y direction during cutting, b) and in that the direct-lightinstrument (7) contains a laser scanning instrument (9), which consistsof two thick glass wedge plates (11 a, 11 b) which are inclined inrelation to the optical axis (8) and can be rotated independently of oneanother about the optical axis (8), and which produce a beam deflectionowing to their wedge angle, the resulting deflection angle α of thelaser beam (18) in relation to the optical axis (8) being variable byrotating the glass wedge plates (11 a, 11 b), c) and in that the laserbeam (18) has a lateral beam offset in relation to the optical axis (8)at the output of the laser scanning instrument (9), owing to thethickness and the oblique setting of the glass wedge plates (11 a, 11b), and it strikes the middle of the objective pupil (19) of theobjective (10) for all deflection angles α.
 2. The laser microdissectiondevice as claimed in claim 1, characterized in that the laser lightsource (5) is a UV laser or an IR laser or a VIS laser.
 3. The lasermicrodissection device as claimed in claim 1, characterized a) in thateach glass wedge plate (11 a, 11 b) is assigned a motor (14 a, 14 b) forrotating the glass wedge plate (11 a, 11 b) about the optical axis (8),and b) in that the motors (14 a, 14 b) are assigned a motor controller(15).
 4. The laser microdissection device as claimed in claim 1,characterized a) in that each glass wedge plate (11 a, 11 b) is assigneda motor (14 a, 14 b) for rotating the glass wedge plates (11 a, 11 b)about the optical axis (8), b) in that the motors (14 a, 14 b) areassigned a motor controller (15), c) in that a computer (26) having amouse and a monitor (28) is provided, the computer (26) being connectedto the motor controller (15) and to the laser light source (5), d) inthat a camera (16) is provided which takes a picture of the preparation(3), said picture being displayed on the monitor (28).
 5. The use of alaser microdissection device as claimed in claim 4, characterized by themethod steps: a) defining a cutting line on the monitor (28) by means ofthe mouse, b) computerized division of the cutting line into a series ofcontiguous cutting holes, the centers of which correspond to thesetpoint positions of the laser beam that are to be occupied on thepreparation (3) during the cutting process, c) calculating thedeflection angle α of the laser beam (18) for each individual positionto be occupied, and calculating the associated rotational settings ofthe glass wedge plates (11 a, 11 b), d) producing the control signalsfor the motorized rotation of the glass wedge plates (11 a, 11 b), e)and producing the defined cutting line by deflecting the laser beam (18)into the calculated setpoint positions by rotating the glass wedgeplates (11 a, 11 b).
 6. The use of a laser microdissection device asclaimed in claim 1, characterized in that the laser beam deflected bythe laser scanning instrument is used for processing of materials. 7.The use of a laser microdissection device as claimed in claim 5,characterized in that the deflected laser beam is guided under computercontrol, and it is used for scribing surfaces.
 8. The use of a lasermicrodissection device as claimed in claim 1, characterized in that thedeflected laser beam is used as optical tweezers, by using it to pick upand transfer individual particles.